Fractionation of sewage sludge colloids has been achieved using the split-flow-thin (SPLITT) fractionation technique. It reveals that the majority of organic carbon as well as heavy metals is carried by particles smaller than 1 μm. Optimization of volumetric flowrates and application of full-feed depletion mode of SPLITT fractionation allows concentrating the smallest colloids. Additionally, HPLC and 13C NMR analyses has been performed to characterize the organic colloids. The 13C NMR spectrum confirms that smaller fraction scontained elevated contents of aliphatic, aromatic and carboxylic groups characteristic of humic compounds. HPLC analysis separates two fractions of substances and compares obtained chromatograms with separation of the humic standard.